Low-level energy laser therapy

ABSTRACT

Methods and systems ( 10, 54, 72 ) are provided for administering phototherapy to an injured tissue in a living subject by delivering visible or infrared biostimulatory energy to bone marrow at a dose sufficient to cause mesenchymal stem cells to appear in the injured tissue. The energy may be coherent light and can be administered transcutaneously, subcutaneously, or via an intramedullary probe ( 74 ). The technique is useful for treating many types of tissue injury, including ischemic cardiac and renal conditions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/179,376, filed May 19, 2009, which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the controlled application of therapeuticlight energy. More particularly, this invention relates to thetherapeutic irradiation of marrow-containing bone using infrared lightand energy of other wavelengths.

2. Description of the Related Art

The meanings of certain acronyms and abbreviations used herein are givenin Table 1.

TABLE 1 Acronyms and Abbreviations ATP adenosine tri-phosphate BM bonemarrow ECHO echocardiographic ICR imprinting control region IR infraredIRI Ischemia reperfusion injury LAD anterior descending branch of leftcoronary artery LED light-emitting diode LLLT low level laser therapy LVLeft ventricle MI myocardial infarction MSC mesenchymal stem cell SEMstandard error of the mean

The bone marrow is a complex tissue featuring several different types ofprimitive cells: hematopoietic stem cells, mesenchymal stem cells(MSCs), endothelial progenitor cells, side population cells, andmultipotent adult progenitor cells. Like other stem cells, mesenchymalstem cells are capable of multilineage differentiation from a singlecell and in vivo functional reconstitution of injured tissues. One ofthe properties of stem cells is their capacity to migrate after infusionto one or more appropriate microenvironments. Certain stem cells areable to exit their production site, circulating in the blood beforereseeding in their target tissues. For mesenchymal stem cells, thenature of homing sites and circulation into peripheral blood is stillunder debate. However, mesenchymal stem cells have been found afterinfusion in multiple tissues, leading to the hypothesis that they canhome, and that they adjust their differentiation pathways to diversetissue microenvironments.

In the last decade cellular therapy for cardiac repair has undergonerapid transition from basic science research to clinical reality. Theapproach to cardiac repair based on stem cells was first realized viaearly studies that induced in-vitro differentiation of stem cells intocardiomyocytes. Orlic et al. first reported that injection of bonemarrow cells with specific markers (Lin⁻/c-kit⁺) to hearts followinginduction of myocardial infarction resulted in reconstitution of 68% ofthe infarcted myocardium, the formation of new blood vessels,improvement in left ventricle function and attenuation of remodeling(Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson S M, Li B, PickelJ, McKay R, Nadal-Ginard B, Bodine D M, Leri A, Anversa P. Bone MarrowCells Regenerate Infarcted Myocardium. Nature. 2001; 410:701-5. Inanother study MSCs were injected intravenously into rat hearts (BittiraB, Shum-Tim D, Al-Khaldi A, Chiu R C. Mobilization and Homing of BoneMarrow Stromal Cells in Myocardial Infarction. Eur. J. CardiothoracSurg. 2003; 24(3):393-8). It was found that labeled cells were seen inand near the infarct up to eight weeks post myocardial infarction, whilenone was seen in sham-operated hearts. It was concluded that followingmyocardial infarction, mesenchymal stem cells are signaled and recruitedto the injured heart, where they undergo differentiation and mayparticipate in the remodeling process.

SUMMARY OF THE INVENTION

In embodiments of the present invention, bone marrow is irradiated invivo in order to treat a variety of disease conditions. The irradiationmay be performed by transcutaneous application of infrared laserradiation over the area of a marrow-containing bone. Alternatively,other radiation sources may be used, including both coherent andincoherent sources, at both IR and other wavelengths. Althoughtranscutaneous irradiation has the advantage of being non-traumatic, theirradiation may alternatively be applied directly to the bone marrowusing invasive techniques.

There is provided according to embodiments of the invention a method ofphototherapy, which is carried out responsively to a determination thatan injured tissue exists in a body of a living subject by irradiatingmarrow within a bone in the body that is remote from the injured tissuewith biostimulatory radiation of sufficient intensity to engender repairof the injured tissue. The bone may be the tibia, although othermarrow-containing bones, e.g., sternum, vertebrae, and pelvic bones mayalso be subjected to the biostimulatory radiation.

According to an aspect of the method, irradiating is performed bydelivering visible or infrared light energy to the marrow by positioninga probe including a source of coherent light on a body surface of thesubject, and transcutaneously directing the energy from the probe towardthe marrow. The source of coherent light may be a laser or alight-emitting diode. The energy may be directed in a continuous wavemode of operation or in a pulsed mode of operation.

According to a further aspect of the method, the injured tissue may becardiac tissue or renal tissue.

According to another aspect of the method, irradiating is performed bydelivering visible or infrared light energy to the marrow by positioninga laser probe beneath a body surface of the subject, and directing theenergy from the probe toward the marrow.

According to yet another aspect of the method, positioning the laserprobe is performed by inserting a distal portion of the probe into amedullary cavity of the bone.

According to an additional aspect of the method, irradiating includespositioning multiple laser probes on the subject and directing theradiation from the multiple laser probes to the marrow simultaneously.

According to still another aspect of the method, the bone includes aplurality of bones, including at least a first bone and a second bone,wherein irradiating includes directing first and second ones of themultiple laser probes to the first bone and the second bone,respectively.

According to yet another aspect of the method, the radiation has awavelength between 630-910 nm.

According to an additional aspect of the method, the radiation has awavelength of 660 nm.

According to a further aspect of the method, the radiation has awavelength between 790-830 nm.

According to still another aspect of the method, the radiation has awavelength of 804 nm at a power of 10 mW/cm2 and an exposure duration of100-120 seconds.

According to a further aspect of the method, a beam diameter of theradiation within the marrow is 0.3 mm.

According to an additional aspect of the method, irradiating isperformed multiple times according to a cyclic dosage schedule. Thecyclic dosage schedule may include six doses administered twice a week.

There is further provided according to embodiments of the invention amethod of phototherapy, which is carried out responsively to adetermination that an injured tissue exists in a living subject bydelivering visible or infrared light energy to bone marrow in a bone ofthe subject at a dose sufficient to cause mesenchymal stem cells toappear in the injured tissue.

There is further provided according to embodiments of the invention anapparatus for administration of phototherapy, including a transparentoutput interface, which is configured to be brought into contact with askin surface overlying a bone in a body of a human subject, and a sourceof coherent light that emits visible or infrared biostimulatoryradiation through the output interface so as to irradiate marrow withinthe bone at a sufficient intensity to engender tissue repair at alocation in the body that is remote from the bone.

An aspect of the apparatus includes a flexibly molded applianceadjustable to fit the body and having a plurality of openings therein,wherein the source of coherent light includes a plurality of probesreceivable in the openings and directed by the openings to emit infraredor visible radiation through the skin surface toward the marrow.

According to a further aspect of the apparatus, the source of coherentlight comprises at least one Gallium Aluminum Arsenide laser.

According to aspect of the apparatus, the radiation has a wavelengthbetween 630-910 nm.

According to one aspect of the apparatus, the radiation has a wavelengthbetween 790-830 nm.

There is further provided according to embodiments of the invention anapparatus for administration of phototherapy, including a flexible probeadapted for insertion into a medullary cavity of a bone in a body of ahuman subject. The probe has a partially cladded distal portion for exitof laser light radially throughout the distal portion for irradiation ofthe medullary cavity, and a source of coherent light, generatingsufficient visible or infrared light energy to stimulate mesenchymalstem cells in the irradiated medullary cavity to facilitate repair ofremotely injured tissue of the subject.

According to still another aspect of the apparatus, the light energy hasa wavelength between 630-910 nm.

According to yet another aspect of the apparatus, the light energy has awavelength between 790-830 nm.

According to a further aspect of the apparatus, the source of coherentlight includes at least one Gallium Aluminum Arsenide laser.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

For a better understanding of the present invention, reference is madeto the detailed description of the invention, by way of example, whichis to be read in conjunction with the following drawings, wherein likeelements are given like reference numerals, and wherein:

FIG. 1 is a pictorial diagram of a light irradiation system, which isconstructed and operative in accordance with an embodiment of theinvention;

FIG. 2 illustrates an appliance for alignment of therapeutic laseroutput with desired locations in the human iliac bone;

FIG. 3 is a pictorial illustration of a light irradiation system, whichis constructed and operative in accordance with an alternate embodimentof the invention;

FIG. 4 is a schematic, pictorial illustration of an intramedullary lightirradiation system, which is constructed and operative in accordancewith an alternate embodiment of the invention; and

FIG. 5 is a flow chart of a method for delivering low-level coherentlight therapy to the bone marrow of a living subject in accordance withembodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the various principles ofthe present invention. It will be apparent to one skilled in the art,however, that not all these details are necessarily always needed forpracticing the present invention. In this instance, well-known circuits,and control logic have not been shown in detail in order not to obscurethe general concepts unnecessarily.

System Architecture

The inventors have found in animal experiments that transcutaneous IRirradiation of the bone marrow is effective in promoting tissue recoveryfollowing myocardial infarction. Without being bound by any particulartheory, the following discussion is offered to facilitate understandingof the invention: It is believed that in vivo irradiation of the bonemarrow at sufficient intensity may stimulate growth of stem cells, whichmigrate to the disease site and participate in repair of the damagedheart tissues. The scope of the present invention, however, is notlimited to treatment of myocardial infarction. Rather, embodiments ofthe present invention may be applied in treating a variety of diseases,irrespective of the precise physiological mechanisms that are stimulatedby bone marrow irradiation.

Recruiting stem cells from autologous BM to targeted ischemic or injuredorgans, e.g., brain or muscles, using the LLLT techniques disclosedherein may inhibit degeneration in diseases such as amyotrophic lateralsclerosis, muscular dystrophy, Parkinson's disease, brain trauma,multiple sclerosis, and stroke. The mechanisms may include replacinglost cells, attenuating degeneration by growth factors that might beproduced by the recruited stem cells or any other type of cells in thebone marrow. As described in U.S. Pat. No. 6,395,016, which is hereinincorporated by reference, the laser may also cause stimulation of theactivity of any type of laser-treated cell, increase its survival afterhoming into the injured site, attenuate the differentiation of any typeof cell to other kind of cell or cause certain cell type to increase therate of secretion of growth factors either in the bone marrow, the bloodor the homing site.

There are also many other degenerative and ischemic diseases andtraumatic conditions that involve loss of function, for example in theliver, kidneys, digestive tract, bones etc. It is shown in theexperiments described below that MSCs stimulated by LLLT home inspecifically on injured/ischemic organs. It is expected that LLLT to theBM may also be helpful in these diseases too.

Turning now to the drawings, reference is initially made to FIG. 1,which is a pictorial diagram of a light irradiation system 10, which isconstructed and operative in accordance with an embodiment of theinvention. A source of coherent light is linked to a control unit 14.The source may be a laser probe 12 as shown in FIG. 1. Alternatively,the other forms of coherent light may be used, e.g., solid state devicessuch as light-emitting diodes (LEDs). The laser probe 12 deliverslow-level light output to a target tissue, details of which arepresented below. The control unit 14 regulates the dosage of the lightoutput as to intensity, duration, and time schedule. While the system 10is shown in FIG. 1 as two separate components, this is exemplary. Thecontrol unit 14 and the laser probe 12 may be integral. For example thesystem 10 may be realized as a single, hand-held device. The laser mayoperate in a pulsed mode or a continuous wave mode of operation. Anenergy dose delivered in pulsed mode penetrates thick bones better andwith less local heating than continuous wave mode for a given poweroutput. Laser output devices described in U.S. Pat. No. 6,395,016 aresutiable for the laser probe 12.

Optionally, the system 10 includes or is linked to a storage device thatholds data generated by the control unit 14. Such data may be used forresearch purposes, to aid treatment analysis, or to support businessrequirements of the therapeutic applications delivered by the system 10.

The target tissue is typically a marrow-containing bony structure. Theexample of FIG. 1 illustrates a transverse section through the shaft oftibia 18. The laser probe 12, shown facing the medial surface of tibia18, is positioned against or proximate skin 20, soft tissues 22 and bonycortex 24, the target tissue being bone marrow tissue in medullarycavity 26. It is recommended, but not essential, to apply energy to bonemarrow tissue having hematopoietic marrow, e.g., bones of the pelvicgirdle or the sternum in adult subjects. When dealing with youngchildren, long bones such as the tibia may be chosen for convenience.The skin of the medial tibia is particularly advantageous as there isrelatively little intervening soft tissue.

Reference is now made to FIG. 2, which schematically illustrates anappliance 28 for alignment of therapeutic laser output with desiredlocations in the human iliac bone. The appliance 28 is shownsuperimposed on anterior and posterior views of an iliac bone 30 in theupper and lower portions of the figure, respectively. The appliance 28can be realized as a mold made of silicon (or similar material) to befitted around the pelvic girdle of a patient. Fixed openings or slots32, 34, 36, 38 in the appliance 28 are adapted to receive respectivelaser probes (not shown) therein and direct the output energy to anintended target. While four slots are shown representatively in FIG. 2,the appliance 28 may have any number of slots. Laser light exiting slots32, 38 may be directed to desired locations on the iliac bone 30, forexample points 40, 42 on the anterior iliac crest, as best appreciatedin the upper portion of FIG. 2. Laser probes in slots 34, 36 may beaimed at points 44, 46 on the posterior iliac crest as shown on thelower portion of FIG. 2.

Those skilled in the art can modify the appliance 28 for application oflaser therapy to other bones, such as the ribs, vertebrae and sternum,all of which normally contain active bone marrow. Indeed, the appliance28 may be elaborated into a body vest suitable for laser application tomany bones simultaneously.

Reverting to FIG. 1, the light application may be non-invasive, orinvasive. In the latter case the geometry of the probe is adapted toinsertion through a skin puncture or incision. Further alternatively,the light may be delivered via a probe or trephine that can penetratethe cortex 24 for application directly into the medullary cavity 26,using known light-handling techniques such as fiberoptics.

A light source may comprise one or more diode lasers 48 disposed in thelaser probe 12. Alternatively, although not illustrated in FIG. 1, thelasers may be disposed in the control unit 14. In the latter case,fiberoptic channels may be incorporated in cable 50 for delivery of theradiation through the probe. The lasers 48 are preferably configured todeliver light in the near infra-red spectrum. However known sources ofcoherent light that emit in the visible spectrum may also be used.

Optionally, the laser probe 12 may be fluid-cooled in order to minimizediscomfort and to avoid the possibility of burning the skin duringapplication. For example, a heat radiator 52 may be provided. Otherknown air or liquid cooling techniques are also suitable.

The following considerations apply in the design of the light deliverysystem:

1. In embodiments employing fiberoptic delivery, the tip of eachfiberoptic channel should have a relatively large diameter (1-2 cm) toavoid heating of the skin upon placement.

2. The laser probe 12 should insure “full contact” between the outputinterface of the probe and the skin. To this end the laser probe 12 maybe constructed by adaptation of the teachings of U.S. Pat. No. 5,149,955and U.S. Patent Application Publication No. 2010/0053391, which areherein incorporated by reference.

3. The apparatus should have the ability to deliver 50 mW to 7 W ofoptical power at the tip of the probe. For non-invasive applications, anoutput power level in excess of 1 W is desirable, in order to deliver anenergy dose of sufficient intensity to engender tissue repair at alocation in the body that is remote from the bone. Delivery of at least10 mW/cm² to the bone marrow is needed for this purpose. Yet, 12-14mW/cm² seems to have a beneficial effect at least as good as 10 mW/cm²When light is radiated directly into the medullary cavity 26, probeswith energy levels at the lower end of the range are sufficient.

4. An emergency “shut off” button should be provided on the laser probe12 or the control unit 14.

5. The lasers 48 may be GaAlAs lasers, for emission at wavelengths ofabout 790-830 nm. Other biostimulatory wavelengths between about 630-910nm can also be used. The radiation output may be either continuous-waveor pulsed.

Alternate Embodiment 1

Reference is now made to FIG. 3, which is a schematic, pictorialillustration of a light irradiation system 54, which is constructed andoperative in accordance with an alternate embodiment of the invention.In this embodiment a radiation delivery unit 56 comprises multiple laserprobes 58. The delivery unit 56 is connected to a suitable energy source60. The probes 58 are spaced apart on the body surface or on the surfaceof a marrow-containing bone 62. An optional mounting plate 64 for theprobes 58 is shown in contact with periosteum overlying cortex 66. Theplate 64 may be suitably fenestrated to accommodate the probes 58.Alternatively, the plate 64 may be and may be solid and transparent tolight at the output wavelength of the probes 58. The plate 64 may beconstructed using flexible materials in order to assure good contactbetween the probes 58 and irregular bone or body surfaces. As discussedabove, a portion of the radiation emitted by the probes 58 penetratesinto marrow cavity 68. In any case, advantages of the system 54 whencompared with a single probe are increased flux of radiation deliveredto a therapeutic radiation field 70 in the marrow cavity 68 and a moreuniform distribution of radiation throughout the field 70.

Therapy may be administered using multiple instances of the deliveryunit 56 simultaneously in the same or different body sites, for exampleone delivery unit 56 on each tibia. Many combinations involvingdifferent bones will occur to therapists skilled in the art.

Alternate Embodiment 2

Reference is now made to FIG. 4, which is a pictorial illustration of anintramedullary light irradiation system 72, which is constructed andoperative in accordance with an alternate embodiment of the invention. Aflexible fiberoptic probe 74 is inserted through a hole 76 that isdrilled through the cortex 66 into the marrow cavity 68. The distalportion of the probe 74 is partially cladded using any suitableincomplete, cladding arrangement, for example having discrete openings,circumferential or longitudinal slots that enable radiation to escaperadially, preferably in all directions from the probe 74 along thelength of the distal portion, as represented by arrows 78. The fiber canbe incorporated in a modified version of the multi-lumen bone marrowaspiration that is taught in U.S. Pat. No. 6,849,051, which is hereinincorporated by reference.

This embodiment has the advantage of delivering energy to the marrowmore efficiently than the non-invasive embodiments described above, butcarries the risk of infection, pain and other possible complications. Asin the other embodiments, the power is adjusted to deliver a desireddose to the marrow cavity 68. The system 72 can operate at lower powerthan the non-invasive embodiments described above.

Operation

Reference is now made to FIG. 5, which is a flow chart of a method ofphototherapy for delivering low-level coherent light therapy to the bonemarrow of a living subject, which can be practiced with any of theabove-described embodiments.

The process begins at initial step 80. It is assumed that the subjecthas been shown to have a medical indication for phototherapeuticstimulation of bone marrow tissue. Conventional preparation of theselected sites is undertaken, according to whether invasive ornon-invasive techniques are to be employed. This may involve, forexample, a surgical incision and perforation of the cortex of a bone inthe case of invasive techniques. Baseline tissue samples for monitoringthe effect of the procedure may be collected.

Next, at step 82 one or more laser probes are applied to each of theprepared sites and connected to a suitable energy source. A cyclicaldosage schedule may be programmed into the control unit of the system totake into account local heating effects and the need to temporarilyinterrupt the energy flow to avoid cellular injury from overheating.Suggested dosage intervals, laser output wavelengths, power levels andcumulative doses are noted above, and described with particularity inExperiments 1, 4 and 8, and elsewhere in the disclosure below.

Next, at step 84 the probes are energized and irradiation is begun. Atimer or other indicator of a termination criterion may be activated,for example a detector of temperature elevation of the probe, tissueheating, or expiration of a time interval.

Then, at delay step 86, it is determined if a termination criterion hasbeen achieved. If so, then at step 88 the probe is de-energized.

At final step 90 the probes are withdrawn from the subject. Another setof tissue samples may be collected at this time, and the processterminates. Generally the process is iterated following injury. Aftereach iteration, the tissue samples may be tested to determine the numberof MSCs using the assay techniques described above. Additionally oralternatively the injured tissue or organ may be subjected to tests todetermine its status responsively to the irradiation. For example, inthe case of myocardial infarction, functional tests such as ejectionfraction determination, normalization over time of electroanatomicparameters, and normalization of blood enzymes may be used as indicia ofthe effectiveness of the irradiation. The dosage intervals and amountsmay be adjusted in accordance with deviations of the one or more of suchtests from expected results.

Various markers may be used for semiquantitative follow up of the extentof the damage to the patient's heart after MI. Several known markers canbe found in the blood. Damage to the heart is correlated with bloodmarker accumulation over time subsequent to MI. Troponin-T or Troponin-Ideterminations in the blood or more precisely their accumulation in theblood during the first week post-MI can serve as an estimate both ofheart damage and an aid to the estimation of the supportive benefitderived from LLLT.

Another marker is the measurement of the amount of the totalantioxidants in the blood of the patients post MI, usually measureddaily. It is known that in dogs post-MI that the level of antioxidantsin the blood is inversely related to the extent of scarring.Measurements of antioxidants in the blood of the patients may also servealso as a useful marker of the effect of the laser treatment, andsupport decisions to vary the schedule of multiple LLLT applicationspost MI. Additionally or alternatively, the commonly usedechocardiographic (ECHO) studies of heart function can be performed inpatients. Comparison of pre- and post-MI ECHO studies prior to andduring the course of LLLT treatments can aid clinical decisionsregarding continuation or variation of LLLT treatments, e.g., bychanging laser output parameters.

Medical Applications

The clinical application of LLLT to humans following myocardialinfarction is expected to be performed as follows: Patient will receivea first laser treatment as described above, followed by multipleadditional laser applications to the BM at intervals as described belowin Experiment 6. Multiple applications are preferred as they arebelieved to maximize migration of stem cells to the injured heart (orother affected sites) following the ischemic injury.

Another indication for treatment using embodiments of this inventionarises in human newborns, who have experienced transient intra-partumischemia for various reasons known in the obstetrical art, withresultant insult to the brain and the likelihood of subsequentneurological impairment. Such newborns may benefit neurologically frommultiple laser applications to the tibia using the method disclosed withrespect to FIG. 5 or as described below in Experiment 7.

Following BM transplantation there might be a delay in the production ofBM cells. It was demonstrated in Experiment 1, described below, thatLLLT enhances the proliferation of MSCs. It can therefore be postulatedthat LLLT to the BM may also enhance the production of various othertypes of marrow cells and the survival of such cells. LLLT mayaccordingly improve function of the immune system and the numbers ofcirculating blood cells in laser-treated patients, especially in theearly post-BM transplantation period.

As a further application for the phototherapeutic techniques disclosedherein, various chemicals, growth factor, and cells other than MSCs thatare stimulated by the treatments and can be isolated from the bonemarrow, blood or lymphatic circulation of experimental animals orpatients may be used as a drug or for cell therapy purposes to mediaterepair or functional improvement of many non-traumatically diseased ortraumatized organs in the body.

Experiments

The following animal experiments were conducted to evaluate treatment ofthe heart.

Experiment 1.

The aim of this experiment was to investigate the effect of laserapplication to bone marrow (BM) in vivo on MSC proliferation capacitywhen cultured in vitro; and the possibility that low level laser therapyapplication to autologous BM can induce and recruit MSCs to theinfarcted rat heart, reduce scarring post myocardial infarction (MI),and stimulate blood vessel formation in hearts following MI.

A total of 24 Charles River male rats weighing 200-250 gm, underwentligation of the anterior descending branch of the left coronary artery(LAD) to induce MI, as described previously (U. Oron, T. Yaakobi, A.Oron, G. Hayam, L. Gepstein, T. Wolf, O. Rubin, and S. A. Ben-Haim,Attenuation of Infarct Size in Rats and Dogs after Myocardial Infarctionby Low-Energy Laser Irradiation. Lasers Surg. Med. 28:204-211.2001).Briefly, rats were anesthetized with Avertin (1 ml/100 g body weightI.P.) and lung ventilated. Thoractomy was performed by incision of theintercostal muscles between the 5th and 6th ribs and the heart wasexposed. The LAD was occluded with 5-0 polypropylene thread (EthiconInc., Cincinnati, Ohio). Following LAD occlusion the thorax and chestwere closed and the rats were ventilated until regaining consciousness.Food and water were supplied ad libitum.

A diode (GaAlAs) laser, wavelength 804 nm with a tunable power output ofmaximum of 400 mW (Lasotronic Inc., Zug, Switzerland) was used. Thelaser device was equipped with a metal-backed glass fiber optic (1.5 mmdiameter). An infrared viewer (Lasotronic Inc. Zug, Switzerland) andinfrared-sensitive detecting card (Newport, Inc., Irvine, Calif.) wereused to determine the infrared irradiation area.

LLLT to the BM was performed by placing the distal tip of an opticalfiber directly on the middle portion of the medial part of the tibiaafter making a small incision in the skin. The beam diameter of thelaser within the bone marrow was 0.3 cm after transmission through themedial part of the tibia. The power of irradiation on the BM was set to10 mW/cm² and the exposure duration was 100 sec (comprising 1 J/cm²).Control rats underwent the same procedure as the laser-irradiated groupbut the laser was not turned on. Control and laser-irradiated rats werechosen at random. The above parameters of laser irradiation were keptconstant in all experiments. Nineteen days post-irradiation, the ratswere sacrificed and the hearts were excised.

BM was removed from laser-treated and sham-treated MI-induced rathearts. MSCs isolation was performed essentially as described by Davaniet al., Circulation 2003; 108[suppl II]:II-253-II-258. Mesemchymalprogenitor cells differentiate into an endothelial phenotype enhancevascular density and improve heart function in a rat cellularcardiomyoplasty model. The BM was removed using a stainless steel rodthat was pushed through the BM cavity. The collected BM was incubated ina shaker with 5 ml medium containing type I collagenase (250 U/ml,Sigma, Israel) for 45 min at 37° C. Cells were then cultured at 1.3×10⁶cm² in Dulbeco Modified Eagle Medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 2 mmol/1 L-glutamine, 100 U/ml penicillin, 100 U/mlstreptomycin (Biological Industries, Israel). Forty-eight hours laterthe medium was replaced to remove non-adherent cells (MSCs are known tobe adherent from the above-noted Davani et al. publication), andthereafter replaced every four days. An identical number of cells wascultured in each culture plate for the laser-treated or controlnon-treated MSCs at this point. The MSCs were then cultured for oneweek, harvested from the culture plates and counted.

As shown in Table 2, the number of cells that were counted in thelaser-treated group seven days after culturing was significantly higher(1.5-fold; p=0.006) compared to cells in the non-laser-treated group.

TABLE 2 No. of cells (10⁻⁵) Control 8.5 Laser-irradiated 12.5

In order to determine the extent of scarring (infarct size) in the heartpost-MI, a cross-section sample (1 mm thick) from the central part ofthe infarcted area was taken from all hearts for histology. Eight-micronparaffin sections were prepared from each tissue sample of each heart.Infarct size and angiogenesis were determined as described previously(T. Yaakobi, Y. Shoshani, S. Levkovitz, O. Rubin, S. A. Ben-Haim and U.Oron. Long-term effect of low energy laser irradiation on infarction andreperfusion injury in the rat heart. J. Appl. Physiol.90:2411-2419.2001; H. Tuby, L. Maltz, U. Oron. Modulations of VEGF andiNOS in the rat heart by low level laser therapy are associated withcardioprotection and enhanced angiogenesis. Lasers Surg. Med.38:682-688.2006), using Masson's trichrome staining. Three observers,blinded to control or treated animals, analyzed the area of theinfarcted hearts. Infarct size was expressed in percentage as the totalinfarct area (in mm²) related to the total area of the left ventricle inthree random cross-sections of each heart, using image analysis software(Sigma Scan Pro, Sigma, St. Louis, Mo.).

It was found that LLLT to the BM caused a significant (p=0.041)reduction of 36% in average infarct size in the infarcted hearts of therats that had undergone BM irradiation as compared to those whose BM wasnot laser-treated (Table 3).

TABLE 3 Infarct Size (%) Control 7.6 Laser-irradiated 4.8

In this experiment, the density of blood vessels in the entire leftventricle including the infarcted area and in the infarcted area alonewas also evaluated. LLLT to the BM area in the tibia did not cause asignificant (p=0.1, 1.6-fold) elevation in the average density of bloodvessels in the non-infarcted portions of the left ventricle in treatedrats as compared to the control group (Table 4). In contrast, LLLT to BMcaused a significant (p=0.014) elevation of 2.8-fold in the averagedensity of blood vessels (in tissue microscopic sections) in theinfarcted area, compared to the control group (Table 5).

TABLE 4 Non-infarcted portion of LV Blood vessels/mm² Control 9Laser-irradiated 14

TABLE 5 Infarcted portion of LV Blood vessels/mm² Control 63Laser-irradiated 175

Kidney and liver tissue samples were also collected from theexperimental rats and processed for histology in order to evaluate theappearance of c-kit positive cells in an uninjured organ in rats thathad undergone LLLT to their BM. C-kit positive cells express a receptorthat is useful as a marker for mesenchymal stem cells. The sectionsunderwent c-kit immunostaining. No elevation of c-kit immunopositivitywas observed in the kidneys or liver of laser-treated rats compared tothe control group.

It is concluded from this experiment that LLLT can induce cultured stemcells from the BM to proliferate at a significantly higher rate thanthey would normally proliferate without laser intervention. It is alsoconcluded that laser application about 20 min post-MI to BM cansignificantly reduce scar formation in the infarcted heart. LLLT to theBM also caused a significant increase in blood vessel density in theinfarcted area compared to blood vessel density found in the infarctedarea in non-treated rats. MSCs, identified as immunopositive c-kitstained cells, which are biostimulated by the laser migrate to theinfarcted heart but do not home in to other injured organs like theliver or kidneys in the same animal.

Experiment 2.

The aim of this Experiment was to further explore the beneficial effectsof LLLT to BM on the infarcted heart and to follow the fate of thelaser-stimulated MSCs from the BM in the heart. This experiment was alsodesigned in order to determine the possible beneficial effects on theinfarcted heart of application of LLLT to both the BM and directly tothe infarcted heart.

A total of 44 Charles River male rats weighing 200-250 gm underwentligation of the LAD as described above in Experiment 1. Following MI,rats were divided randomly into four groups: 15 rats served as a sham,non-laser-treated control group; 11 rats were used as a laser-treatedgroup, with the laser applied to the infarcted myocardium, as describedin the above-noted Oron et al. publication. Eight rats were used as alaser-treated group in which the laser was applied to the BM in thetibia; and ten rats received laser irradiation both to the BM in thetibia and to the infarcted myocardium. Control and laser-irradiated ratswere chosen at random after the induction of MI. Laser irradiation tothe BM was performed as described in Experiment 1. LLLT was performedabout 20 minutes post-induction of MI to the rats. In the hearts thatwere laser-irradiated on the infarcted area, laser irradiation wasperformed by placing the distal tip of a fiber optic probe 4.7 cm fromthe heart. The beam diameter at the heart was 1.8 cm, so that the totalarea of the anterior surface of the heart was laser-treated. Power ofirradiation was set to 12 mW/cm² and exposure duration was 100 sec (1.0J/cm²). Rats were sacrificed 19 days post induction of MI. Sections ofrat hearts were stained with Masson's trichrome for infarct sizedetermination as described in Experiment 1.

LLLT to the infarct caused a significant (p=0.001) reduction of 39% inthe infarct size compared to control. Laser irradiation to the BM causeda significant (p<0.001) reduction of 79% in infarct size, while in thedual-irradiated rats (on both the BM and the infarcted area in theheart) irradiation caused a significant (p<0.001) reduction of 72% inthe infarct size as compared to the control infarcted(non-laser-treated) group. BM (tibia) irradiation showed a significant(p<0.01) reduction of 63% in infarct size and dual irradiation ofinfarct and BM showed a significant reduction (p<0.01) of 54% comparedto infarct irradiation alone. No significant reduction in infarct sizewas observed between the BM of laser-treated rats and that of thosesubjected to dual irradiation. These results are summarized in Table 6,in which the results are Results are mean±SEM of 8-15 rats in eachcolumn.

TABLE 6 Effect of LLLT on infarct size Infarct Size (% of LV) Control 35Laser-irradiated heart 22 Laser-irradiated BM 8 Laser-irradiated BM andheart 10

Irradiation of the BM in the tibia of infarcted rats caused asignificant (p<0.013) elevation of 2.5-fold in the density of c-kitimmunopositive cells in the left ventricle area as compared to thecontrol non-laser-treated rats. Dual laser irradiation to both the BMand the infarcted heart caused a significant (p=0.019) elevation of3.5-fold in the c-kit positive cell density compared to the control. Noelevation in the density of c-kit immunopositive cells was observed inthe group that received laser treatment only to the infarcted heart ascompared to the control (Table 7). No significant differences inimmunopositive c-kit cells were observed between BM irradiation alonecompared to dual irradiation of infarcted zone and BM of the tibia.

TABLE 7 Cells/mm² in LV Control 2 Laser-irradiated heart 4Laser-irradiated BM 5 Laser-irradiated BM and heart 7

TABLE 8 Cells/mm² in infarcted area Control 2 Laser-irradiated heart 13Laser-irradiated BM 44 Laser-irradiated BM and heart 27

In the area of the infarcted zone only the BM irradiation caused asignificant (p=0.05) elevation of 22-fold in the density of c-kitimmunopositive cells as compared to the control group. Dual laserirradiation to the BM and infarcted zone caused a significant (p=0.001)elevation of 13.5-fold in the density of the density of c-kit positivecells as compared to the control. In the hearts that received laserirradiation on the infarcted zone there was a significant 6.5-fold(p=0.016) elevation in c-kit immunopositivity compared to control (Table8). No significant difference was observed in the density of c-kit inthe dual irradiation (on the infarct and BM) compared toBM-laser-treated or infarct laser-treated rats alone and BM irradiationcompared to infarct irradiation alone.

C-kit immunopositivity was significantly (p=0.01) higher (2.6-fold) inthe BM that was laser-irradiated compared to laser treatment of theinfarcted heart. No significant difference was observed in the densityof c-kit in the dual irradiation (on the infarct and BM) compared toBM-laser-treated or infarct laser-treated rats alone, and in BMirradiation compared to infarct irradiation alone.

Left ventricle dilatation (volume of the left ventricle relative to theheart's total volume) is another marker that correlates with reductionin functional performance of the heart. Left ventricle dilatation wascalculated in the present experiment from the microscopic slide sectionsof the heart in the region of the myocardial infarction. The results arepresented in Table 9. The findings indicate that in thenon-laser-treated MI-induced hearts the dilatation value was 27%,indicating dilatation of the left ventricle relative to the intact heartwith no MI. In the rats with MI that were laser-treated to the BM, themeasured value of ventricular dilatation was significantly lower (7%),comprising a reduction of 74% relative to those rats with MI that didnot receive the LLLT.

TABLE 9 Dilatation (% of LV) Control 27 Laser-irradiated heart 19Laser-irradiated BM 7 Laser-irradiated BM and heart 16

It is concluded from this experiment that LLLT applied to BM ofinfarcted rat hearts can reduce the formation of scarring post-MI by80%, while the application of LLLT to the heart directly reducesscarring to about 60% relative to non-laser-treated infarcted rats. Theapplication of LLLT simultaneously to BM and heart does not giveadditional benefit over application to BM only. Significantly more MSCswere found in the infarcted hearts of rats that underwent LLLT to theirBM than those found in the hearts of non-laser-treated rats. It is alsoconcluded from this experiment that LLLT to BM after MI significantlyimproves functional performance of the heart, as indicated by a decreasein the dilatation of the left ventricle.

Experiment 3.

This experiment was designed to investigate the long-term safety oflaser application to the BM on the histology of the BM, liver andkidneys of ICR mice that received laser treatment at several differentdoses and frequencies compared with two control sham-treated groups ofmice.

The laser used in this experiment for all groups was the same as thatdescribed for Experiment 1. The mice groups were as follows:

Group 1 (n=11)—Control mice, anaesthetized only once with the laserplaced on the tibia but not turned on.

Group 2 (n=10)—Control mice, anaesthetized a total of 6 times, twice aweek for 3 weeks. Like Group 1 (above) the laser was not turned on.

Group 3 (n=11)—Laser-treated group. Laser applied only once to the BM inthe tibia to deliver 10 mW/cm² power density to the BM for 2 min.

Group 4 (n=5)—Laser-treated group. Laser applied as in Group 3 (above),but for 6 times, twice a week for 3 weeks.

Group 5 (n=5)—Laser-treated group. Laser applied to deliver a dose of 50mW/cm² for 2 min to the BM only once.

Mice were held under optimal conditions (food and water ad libitum) atroom temperature (22-24° C.) for 7-8 months (about 80% of theirlifespan) and then sacrificed. The BM, liver and kidneys were fixed informalin and processed for histology. After staining withhematoxylin-eosin sections, these organs were viewed under themicroscope. In the livers of both the control non-laser-treated groupand laser-treated group small necrotic centers were noticed, probablydue to aging. No histological differences were observed in the kidneysof the laser-treated mice as compared to the controls. In thehistological sections of the BM, there was no difference between thecontrol and the laser-treated mice, except for a slight elevation inmegakaryocyte density in laser-treated Group 4.

Experiment 4.

In this experiment precise measurements of laser irradiation wereperformed on the tibias of human cadavers to determine the appropriatedose of laser energy to be delivered to the BM in order to achieve atherapeutic effect. The laser used was an infrared laser, wavelength810-830 nm with power output of 400 mW. Measurements of the power of thelaser and its dispersion were made using the equipment described inExperiment 1. Referring again to FIG. 1, the transmission of laserenergy from the laser probe 12 to the medullary cavity 26 was 2%. Theoptimal power density to achieve a therapeutic effect (as described inExperiments 1 and 2) is 12 mW/cm² applied to the BM. Moreover, the laserbeam diverged during its passage through the bone from 4 mm diameter atthe tip of the fiber optic on the skin to about 20 mm in the medullarycavity 26. In order to correct for these effects, the laserconfiguration in the laser probe 12 equivalent to a single laser havinga beam diameter of 2 cm is required at the laser probe 12 in order toavoid local heating due to high power output. The power output from aprobe operating in the configuration of FIG. 1 was about 1.6 W.

Experiment 5.

This experiment was designed to determine whether LLLT applied to the BMfour hours post-MI would yield a beneficial effect, measured byreduction of scarring relative to non laser-treated rats and incomparison to LLLT applied immediately post-MI as described inExperiments 1 and 2 above.

Twenty-three rats post-MI were used in this experiment. Eight ratsreceived LLLT application to the bone marrow four hours post-MI, whilethe rest (n=15) served as sham-operated MI-induced controls. The laserparameters and application was identical to those described inExperiments 1 and 2 above. The rats were sacrificed 21 days post-MI andtissue cross-sections from the heart, including the infarcted area, wereprocessed for histology. The histological sections were also stainedusing c-kit, and quantitative analysis was performed for determinationof infarct size, i.e., the extent of scarring and dilatation asdescribed in Experiments 1 and 2.

Table 10 demonstrates that laser treatment to the BM caused asignificant reduction in infarct size (52%; p=0.01) compared to thecontrol (non-laser-treated) group.

TABLE 10 Infarct Size (%) Control 25 Laser-irradiated 12

Dilatation measurements, shown in Table 11, were calculated as the ratioof the area of the left ventricular cavity to the total area of the leftventricle, using randomly selected tissue sections from the infarctedarea. The results show that dilatation in the laser-treated group wassignificantly smaller (42%; p=0.018) than in the control group.

TABLE 11 Dilatation (% of LV) Control 33 Laser-irradiated 19

Immunostaining for c-kit, shown in Table 12, indicate that in the entireLV area of laser-treated hearts there was a significantly higher c-kitimmunopositive cell density (3.8-fold; p<0.001) compared to thenon-laser-treated-group. In the infarcted zone, shown in Table 13, therewas a significantly higher cell density (4.2-fold; p<0.001) of c-kitimmunopositive cells than in the laser-treated group compared to thecontrol.

TABLE 12 Cells/mm² (in LV) Control 4 Laser-irradiated 16

TABLE 13 Cells/mm² (in infarcted area) Control 4 Laser-irradiated 19

In conclusion, this experiment demonstrates that even when lasertreatment is applied 4 hours post-MI, there is a significant beneficialeffect on the infarcted rat heart over non-treated rats. These resultsare of clinical significance, allowing treatment to patients within areasonable “therapeutic window” during which the patient reaches thehospital and MI is diagnosed.

Experiment 6.

This experiment is similar to Experiment 5, but the rats were left for 6weeks until sacrificed. In this experiment MI was produced in 12 ratsusing the techniques described above. Nine of them were laser-irradiatedfour hours after MI was induced and the other three were sham-operated.Rats were sacrificed six weeks post-MI. Bone marrow was extracted inorder to measure the number of MSCs in the bone marrow 6 weeks postirradiation, and histological sections were taken.

The results indicate no difference in the number of MSCs in BM extractedfrom the irradiated tibia in the laser-treated group compared to thecontrol 6 weeks post-irradiation (data not shown). Infarct size, shownin Table 14, was significantly smaller (50%; p=0.041) in thelaser-treated compared to the non-laser-treated group. Dilatation wasnot significantly less (32%; p=0.09) in the laser-treated group comparedto control, as shown in Table 15.

TABLE 14 Infarct Size (%) Control 14.2 Laser-irradiated 7

TABLE 15 Dilatation (% of LV) Control 19 Laser-irradiated 13

C-kit staining showed a significantly higher density of MSCs (3-fold;p=0.03) in the infarcted zone in the laser-treated group compared to thecontrol group, as shown in Table 16. There was no statistical differencein the MSC density of the entire LV between the laser-treated group andthe control group.

TABLE 16 Cells/mm² (infarct) Control 1 Laser-irradiated 3Experiment 7.

This experiment was designed to determine whether LLLT applied to BM at7 days post-MI, followed by multiple applications of LLLT at 14 and 21days post-MI, would yield a beneficial effect (reduction of scarring)relative to non-laser-treated rats and in comparison to LLLT appliedimmediately as described in Experiments 1 and 2.

MI was induced in 23 rats as described above for this experiment. Nineof them received LLLT to the bone marrow seven days post-MI while theothers served as sham-operated MI-induced rats. The laser parameters,application, and evaluation techniques were identical to those describedin Experiments 1 and 2. Two additional irradiations were applied on days14 and 21 post-MI. The rats were sacrificed on day 28. The control groupwas sham-operated and sacrificed on the same day as the treated group.Tissue sections from the infarcted hearts were processed for histology.The histological sections were stained for c-kit, and quantitativeanalysis was performed for determination of infarct size (extent ofscarring) and dilatation as described in Experiment 1.

There was a significantly higher reduction (57%; p=0.004) in the infarctsize from 26% in the non-LT-treated rats to 11% in the LT rats (Table17). Dilatation was significantly less (36%; p=0.027) in thelaser-treated group compared to non-laser-treated controls (Table 18).Moreover, a significant (P=0.004) 2.9-fold increase occurred in thenumber (per area) of c-kit positive cells in the LV area (Table 19) anda significant (p<0.001) 5-fold increase in c-kit in the infarcted area(Table 20) of the LT rats as compared to the controls.

TABLE 17 Infarct Size (%) Control 26 Laser-irradiated 11

TABLE 18 Dilatation (% of LV) Control 33 Laser-irradiated 21

TABLE 19 Cells/mm² (in LV) Control 5 Laser-irradiated 14

TABLE 20 Cells/mm² (in infarcted area) Control 4 Laser-irradiated 18

It is concluded that LLLT applications beginning even a week post-MIwith multiple applications thereafter can cause a significantattenuation in scar formation and ventricular dilatation in theinfarcted heart. This fact is of clinical significance, and promotesLLLT as a possible treatment for patients with existing post-MIscarring.

Experiment 8.

An experiment was performed with infarcted rats to investigate thelong-term effect of LLLT to BM in vivo on MSCs proliferation capacitywhen cultured in vitro three and six weeks post laser application.

A total of 20 Charles River male rats (weight 200-250 g) underwentligation of the left anterior descending artery to induce myocardialinfarction (MI) in the manner described above. The rats were thendivided randomly into two experimental groups. Group I contained 12rats: six were laser-treated while the other six (control) did notreceive laser treatment. Rats in this group were sacrificed three weekspost-MI. In group 2, four rats served as the laser-treated group andfour as a control group. Laser application was carried out 0.5 hours tofour days post-MI. These rats were sacrificed six weeks post-MI. BM wasremoved from both laser-treated and sham-treated tibias of MI-inducedrat hearts. MSCs were isolated, incubated for one week, harvested fromthe culture plates and counted. As shown in Table 21 the number ofcultured MSCs that originated from rats three weeks post-laser treatmentwas significantly higher (1.5-fold; p=0.006) compared with the number ofcells that were grown post-isolation from BM. In the 6-week group therewas no significant difference between the number of MSCs isolated andgrown in culture in the laser-treated or non-laser-treated BM.

TABLE 21 Cells/mm² 3 weeks Control 9 Laser-irradiated BM 13 6 weeksControl 8 Laser-irradiated BM 9

In conclusion, these results indicate that three weeks post-irradiationthere is still a stimulatory effect of laser irradiation on stem cellsin the bone marrow. But six weeks later, the stimulatory effect of thelaser treatment on MSCs isolated from the bone marrow no longer exists.Thus the effect of LLLT on MSCs derived from the tibia bone marrow istransient.

Experiment 9.

This experiment was designed to estimate the number of total c-kitimmunopositive cells in the heart of non laser and laser-treated BM inthe rats based on observations from the rats in Experiment 8.

The volume of the rat heart was estimated to be about 1 cm³ containing10¹⁰ cells of all types. The total number of c-kit positive cells in thelaser-treated rats was estimated (based on estimates of heart and tissuesection volumes and configurations) to be 1,558,700 cells three weekspost MI. This number was calculated based on average cell number inmicroscopic sections of the heart of rats as described in Experiment 1.This number was multiplied by the total number of sections in the rathearts. The number of c-kit positive cells decreased to 58,000 cells inthe laser-treated rats at six weeks post-MI. Thus, if it is assumed thatthe loss of c-kit positive cells is linear from three to six weeks everyweek, there is a loss of 510,230 MSCs. In the group of rats wheremultiple laser applications were applied post-MI (Experiment 7) theestimated number of c-kit positive cells was found to be 1,316,000cells. The interpolated estimated number of MSCs remaining in the heartat four weeks according to the above assumption of loss (510,230 cellsper week) is about 1,078,000 cells. Thus, the number of c-kit positivecells found at four weeks post-MI with multiple laser application isgreater than this estimated number (1,316,000 cells as compared to1,078,000).

The number of MSCs as determined by c-kit positivity found in the heart3 weeks post MI suggest that stem cells migrating to the infarcted hearteven in the laser-treated rats are too few to compensate for the stemcells that were lost as a result of the MI. The number of MSCs decreasedeven further between three and six weeks post MI. As noted above in thesection entitled “Medical Applications”, the data suggest that multiplelaser applications post-MI to the BM may be most likely to achievecardiac functional improvement in recovering post-MI patients.

Experiment 10.

The aim of this experiment was designed to investigate the possibleeffect of LLLT on the process of formation of adhesions and scar tissuein the area of surgical intervention post surgical procedures.

Twelve rats were used for this experiment. All rats underwent surgicalprocedure to produce a temporary occlusion of the right renal artery for15 minutes (three control and five laser-treated rats) or 30 minutes(two controls and two laser-treated) post-renal artery occlusion. Theexposure of the kidney was performed by lateral excision in the skin andmuscles above the kidney area. Seven rats were treated with LLLT to theBM as described in Experiment 1 at 15 minutes after temporary occlusionof the renal artery, and once again a week later. Sham-treated rats usedas controls underwent the same procedure as the laser-treated rats butthe laser was not turned on.

The rats were sacrificed 2 weeks post-surgery. Scarring and adhesions inthe operative site around the right kidney were examinedmacroscopically. While in the non laser-treated rats scarring around thekidney and adhesion of the kidney to adjacent tissues (abdominalmuscles, liver, etc.) were noticed, there was a marked reduction inscarring and adhesions around the right kidney of the laser-treatedrats.

It is suggested that LLLT to the bone marrow may induce proliferation(see Experiment 8) and activation of stem cells or other cells in thebone marrow so that they may migrate to the surgical or traumatized area(via circulating blood) and attenuate the process of scarring, whichresults in a marked reduction of post traumatic adhesions. Thisphenomenon is of clinical importance in any surgical procedure in humanswhere adhesion post surgery may have adverse effects to the body.

Experiment 11,

Renal dysfunction occurs in a clinical condition known as nephriticsyndrome, which may present as total or partial renal impairment. Suchimpairment may result from various clinical conditions. The abovecondition may involve various histological phenomena in various parts ofthe kidney structures (cortex, nephrons, papillary tubules etc.).Ischemic-reperfusion injuries to the kidney in experimental animals maymimic pathological stages of nephritic syndrome in humans.

The aim of this experiment was designed to investigate the possibleeffect of LLLT on ischemia-reperfusion injury (IRI) in the kidney.Twelve rats were used for this experiment. All rats underwent surgicalprocedure to occlude the right renal artery for 15 minutes (threecontrol rats and five laser-treated rats) or 30 minutes (two controlrats and two laser-treated rats (prior to renal artery occlusion). Theexposure of the kidney was performed by lateral excision in the skin andmuscles above the kidney area. Seven rats were treated with LLLT to theBM as described in Experiment 1 at 15 minutes post surgery and onceagain a week later. Sham-treated rats used as controls underwent thesame procedure as the laser-treated rats but the laser was not turnedon.

The rats were sacrificed 14 days post-IRI and tissue cross-sections fromthe right and left kidneys were processed for histology. Thehistological sections were stained for hematoxylin-eosin. Sections wereanalyzed by light microscopy. In the 15 min IRI experiment in thecontrol group kidney sections, section the papilla and the medullarypart of the kidney had marked histopathological damage. The papilla wasdiminished and in the medulla there was significant tubular necrosis. Inthe LLLT sections from rats exposed to LLLT, there was very light injuryin the papillary area, while the medulla and the cortex did not show anydamage. In the rats experiencing IR injury as a result of occlusion for30 minutes, the control group showed no papilla, and displayedsignificant increase in necrosis, large with tubular dilatation in themedulla and cortex, and visible damage in the glomeruli. The sectionsfrom the group treated with LLLT showed small injured papillae andnecrosis and dilatation of the tubules in the medulla and the cortex.

The 15 min IRI sections were also stained for c-kit. The results showedthat in the laser-treated group there was a significant (p=0.02)3.87-fold higher c-kit immunopositive cells per mm² of tissue sectioncompared to the control group.

It is concluded that LLLT to the BM immediately and 1 week post renalIRI prevents necrosis in the papilla, medulla and cortex and preservesthem better when compared with a sham-treated control group.

Experiment 12.

The aim of the Experiment was to further explore the beneficial effectsof diode laser application at a wavelength of 660 nm (as opposed to 810nm in Experiment 2) to BM on the scar formation following MI in the ratmodel.

A total of 12 male Wistar rats weighing 200-250 g underwent ligation ofthe LAD as described in Experiment 1. The rats were divided after MIinto two groups: eight rats served as sham-treated rats served as acontrol group, and four rats were used as a laser-treated group. Controland laser-treated rats were chosen at random after the induction of MI.Laser irradiation to the BM was performed as described in Experiment 1using a diode laser, wavelength 660 nm with a tuneable power output,maximally 40 mW (Lasotronic Inc., Zug, Switzerland). The laser devicewas equipped with a metal-backed glass fiberoptic interface (1.5 mmdiameter). An infrared viewer (Lasotronic Inc. Zug, Switzerland) andinfrared-sensitive detecting card (Newport, Inc., Irvine, Calif.) wereused to determine the infrared irradiation area in the BM aftertransmission through the medial part of the tibia (as described inExperiment 1).

LLLT was applied to the BM about 20 min post induction of MI. The powerof laser irradiation on the BM was set to 10 mW/cm² and the exposureduration was 100 sec (comprising 1.0 J/cm²). Rats were sacrificed 21days post induction of MI. Sections from rat hearts stained with.Masson's trichrome were analyzed for infarct size determination (asdescribed in Experiment 1).

The laser irradiation on the BM caused a significant (p<0.001) reductionof 78% in the infarct size as compared to the control(non-laser-treated) group.

It can be concluded that diode laser application at a wavelength of 660nm to the BM in rats post MI results in a significant reduction of scarformation. It is also concluded that LLLT application to the BM causedbeneficial effect to the IRI kidney in conjunction with significantlygreater c-kit immunopositive cell recruitment, similar to the infarctedheart

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed hereinabove. Rather, the scope of the present inventionincludes both combinations and sub-combinations of the various featuresdescribed hereinabove, as well as variations and modifications thereofthat are not in the prior art, which would occur to persons skilled inthe art upon reading the foregoing description.

The invention claimed is:
 1. A method of phototherapy, comprising thestep of: responsively to a determination that an injured tissue existsin a body of a living subject, irradiating marrow within a bone in thebody that is remote from the injured tissue with biostimulatoryradiation of sufficient intensity to engender repair of the injuredtissue, wherein the subject is an adult or a child; and after performingthe step of irradiating marrow, testing the injured tissue to determineits status responsively to irradiation.
 2. The method according to claim1, wherein the irradiating is performed by delivering visible orinfrared light energy to the marrow by: positioning a probe comprising asource of coherent light on a body surface of the subject; andtranscutaneously directing the energy from the probe toward the marrow.3. The method according to claim 2, wherein the source of coherent lightis a laser.
 4. The method according to claim 2, wherein the source ofcoherent light is a light-emitting diode.
 5. The method according toclaim 2, wherein directing the energy from the probe is performed in acontinuous wave mode of operation.
 6. The method according to claim 2,wherein directing the energy from the probe is performed in a pulsedmode of operation.
 7. The method according to claim 1, wherein theinjured tissue is cardiac tissue.
 8. The method according to claim 1,wherein the injured tissue is renal tissue.
 9. The method according toclaim 1, wherein irradiating is performed by delivering visible orinfrared light energy to the marrow by: positioning a laser probebeneath a body surface of the subject; and directing the energy from theprobe toward the marrow.
 10. The method according to claim 9, whereinpositioning the laser probe comprises inserting a distal portion of theprobe into a medullary cavity of the bone.
 11. The method according toclaim 1, wherein irradiating comprises positioning multiple laser probeson the subject and directing the radiation from the multiple laserprobes to the marrow simultaneously.
 12. The method according to claim11, wherein the bone comprises a plurality of bones, including at leasta first bone and a second bone, wherein irradiating comprises directingfirst and second ones of the multiple laser probes to the first bone andthe second bone, respectively.
 13. The method according to claim 1,wherein the radiation has a wavelength between 630-910 nm.
 14. Themethod according to claim 1, wherein the bone is a tibia.
 15. Anapparatus for administration of phototherapy, comprising: a transparentoutput interface, which is configured to be brought into contact with askin surface overlying a bone in a body of a human subject, wherein thesubject is an adult or a child; and a source of coherent light, which isconfigured to emit visible or infrared biostimulatory radiation throughthe output interface so as to irradiate marrow within the bone at asufficient intensity to engender tissue repair at a location in the bodythat is remote from the bone.
 16. The apparatus according to claim 15,further comprising a flexibly molded appliance adjustable to fit thebody and having a plurality of openings therein, wherein the source ofcoherent light comprises a plurality of probes receivable in theopenings and directed by the openings to emit infrared or visibleradiation through the skin surface toward the marrow.
 17. The apparatusaccording to claim 15, wherein the source of coherent light comprises atleast one Gallium Aluminum Arsenide laser.
 18. The apparatus accordingto claim 15, wherein the radiation has a wavelength between 630-910 nm.19. The apparatus according to claim 15, wherein the radiation has awavelength between 790-830 nm.